Polylactide cell culture containers and use in cell culture

ABSTRACT

2D and 3D cell culture containers formed from blends of poly-L-lactide and poly-D-lactide provide growth surfaces for adherent cells and do not require surface treatment or coating to support mammalian cell growth. The cell culture containers are transparent, heat tolerant, and are environmentally degradable and suitable for composting in landfills.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation in part of U.S. Pat. No. 11,279,909,which was filed on 6 Dec. 2019 and issued on 22 Mar. 2022, which ishereby incorporated by reference in its entirety.

BACKGROUND

Pre-sterilized disposable plastic cell culture containers have becomeubiquitous for culturing of eukaryotic and prokaryotic cells. Theseplastic cell culture containers are usually fabricated from syntheticpolymer resins including polystyrene, polyvinyl, polycarbonate, andpolyolefin-type (polyethylene and polypropylene) thermoplastics. Onemajor problem arising from the ubiquitous and large-scale use of plasticcell culture containers is that of environmental sustainability.Commercial sterilization of such thermoplastic products is routinelyaccomplished using either gamma irradiation, electron-beam irradiation,or ethylene oxide treatment. With few exceptions, plastic cell culturecontainers are difficult or impossible to reuse because they wouldrequire washing and re-sterilization, in which generally availablelaboratory sterilization equipment (e.g., steam autoclaves) would meltor severely distort most of the conventionally used plastics describedabove that have relatively low softening and melting temperatures.Consequently, these plastic cell culture containers are usuallydiscarded in landfills or destroyed by incineration after use. Landfilldisposal is undesirable because petroleum-derived synthetic plastics donot readily decompose or compost, and incineration can produce toxicgases. Thus, there is an urgent need for cell culture containers thatare environmentally degradable and suitable for landfill compost ordisposal.

When most types of conventional cell culture vessels are used to growanchorage-dependent animal or plant cells (also referred to asattachment-dependent or adherent cells), an interior surface of theculture vessel has to be pre-treated, modified, or coated to promotecellular attachment, without which many cell types will not grow. Whilean untreated polystyrene Petri dish may be useful for culturing bacteriaand yeast cells on a solid nutrient agar, for example, the untreatedpolystyrene dish is not a useful or functional container for growinganchorage-dependent mammalian cells. Various surface treatments andcoatings for promoting cellular attachment to polystyrene cell culturecontainers are known. Such treatments, coatings, or modifications cansignificantly add to the cost and complexity of producing a cell culturecontainer. The cost can rapidly increase for large 2D growth surfaces orfor complex 3D growth surfaces. Thus, there is a need for cell culturecontainers that do not require surface treatments, coatings, ormodifications for successful cultivation of anchorage-dependent animalor plant cells, while still providing cellular attachment and robustcell anchorage.

SUMMARY

The present technology provides cell culture containers that can beutilized for cultivating prokaryotic or eukaryotic cells, includingadherent cells, such as adherent mammalian cells, without requiringsurface treatment of the containers after they have been molded fromthermoplastic resin. Growth surfaces of the cell culture containers ofthe present technology can be untreated and uncoated, or optionally canbe chemically or physically treated or coated to enhance cell adhesion.The cell culture containers disclosed herein can more easily andeconomically have large growth surfaces and/or complex or 3D surfaceshapes because no treatment or coating step is necessary. Further, thecell culture containers of the present technology can be environmentallydegradable and suitable for composting in landfills.

A preferred thermoplastic resin for use in the present technology is ablend of poly-L-lactide resin (PLLA or PLA) and poly-D-lactide resin(PDLA or PDA). PLA can be commercially produced from agriculturalbiomass materials that provide plant starch, and PLA is consideredenvironmentally sustainable. PLA is a polymer of L-lactate monomers, andboth the polymer and monomer are enzymatically degraded and consumed bya variety of microorganisms. PDA is the enantiomer of PLA. PDA is apolymer of D-lactate monomers, which are produced by certain bacteria.Polymerization of D-lactate can produce PDA in various molecularweights.

Formula 1 depicts L-(+)-lactic acid, also known as (S)-lactic acid.

Formula 2 depicts D-(−)-lactic acid, also known as (R)-lactic acid.

To allow visual and microscopic monitoring of cells as they are beingcultured, the cell culture containers can be fully transparent atvisible wavelengths of light. Unfortunately, transparent PLA (purepoly-L-lactide) has a low softening temperature (about 50° C.) thatlimits its use and would be inconsistent with most cell cultureapplications. The addition of microparticulate minerals such as talc andother substances is known to promote PLA crystallization, resulting inhigher softening and melting temperatures. However, the resulting lossof transparency makes microparticle-modified PLA generally unsuitablefor cell culture containers. Methods of admixing PDA resin with PLAresin disclosed herein can provide higher softening and meltingtemperatures while retaining transparency suitable for cell cultureapplications. Annealing methods are disclosed herein which can retainthe transparency of a mixture of PLA and PDA, and the mixture cansimultaneously have a softening temperature greater than 50° C.

A general structure of poly-lactide (or polylactide) is shown in Formula3. In Formula 3, the methyl groups are each attached to a chiral center.Each chiral center can be the (S)-configuration or the(R)-configuration. When referring to a monomer of lactic acid (lactide),the (S)-configuration is (L) or levorotatory. PLA contains lactides inthe (S)-configuration. By comparison, PDA contains lactides in the(R)-configuration which is (D) or dextrorotatory. In Formula 3, themethyl groups can assume either the (S) or (R) configuration, so thegeneral structure of poly-lactide can comprise either enantiomer oflactic acid.

Depending on polymerization conditions, polymerization of either (R) or(S) lactic acid can result in polymers having different molecularweights. For example, a linear molecular formula of poly-lactide can beillustrated with H(C₃H₄O₂)_(n)OCH₃, (C₃O₂H₄)_(n), CH₃(C₃O₂H₄)_(n)CH₃, orwith (C₃O₃H₅)—(C₃O₂H₄)_(n)—(C₃O₂H₅). In Formula 3, for example, witheach monomer unit having a molecular mass of 72 g per mole, a value of“n” greater than 1500 can represent a polymer with molecular weight(M_(w)) greater than 100,000 Da. The value of n selected forpolymerization and resin manufacture will depend upon the polylactideproduct application, where an extruded film may require an n ofapproximately 4,000 or a M_(w) of ≈300,000 Da while an injection-moldedcontainer may require an n of approximately 1,400 or a M_(w) of ≈100,000Da. In general, the value of n can be at least 100, 200, 300, 500, 1000,1400, 1500, 2000, 3000, 4000, or 5000 or more. Optionally coupled withany of these lower limits, as appropriate, the value of n can be lessthan or equal to 10000, 5000, 3000, 2000, 1500, 1200, 1000, or 500.

Polymerization of a racemic mixture of L- and D-lactides can lead to thesynthesis of poly-DL-lactide (PDLLA) polymer, which can be amorphous.Depending on the ratio of enantiomers used during polymerization,different forms of PDLLA can be obtained. Poly(L-lactide-co-D,L-lactide)(PDLLA) is a thermoplastic aliphatic polyester synthesized from bothenantiomers (D and L) of lactic acid. PDLLA has some monomers withalternating (R) and (S)-configuration, as shown in the example monomer‘m’ of Formula 4. In monomer ‘m’ in Formula 4, the leftmost methyl groupis in the (R) (or D) configuration, with the next methyl group in the(S) (or L) configuration. In general, the values of m and n each can beindependently at least 100, 200, 300, 500, 1000, 1400, 1500, 2000, 3000,4000, or 5000 or more. Optionally coupled with any of these lowerlimits, as appropriate, the value of m and n each can be independentlyless than or equal to 10000, 5000, 3000, 2000, 1500, 1200, 1000, or 500.

Different forms of poly-lactide, for example, comprising differentrepeating sequences of D and L in a poly-lactide polymer can yieldmaterials with different degrees of crystallinity, thermal transitions,solubility, and rates of degradation.

The present technology can comprise different forms of poly-lactidepolymer. The molecular weights of the various poly-lactide polymers canvary. The present technology can utilize poly-lactide polymers withbranching at any position, with branching upon branching, or withoutbranching (straight chain polymers).

As used herein, “poly-L-lactide”, “poly(L-lactide)”, “PLA”, or “PLLA”refers to a polymer consisting of (S)-lactic acid and does not refer toracemic PLLA (racemic poly-L-lactic acid). As used herein,“poly-D-lactide”, “poly(D-lactide)”, “PDA”, or “PDLA” refers to apolymer consisting of (R)-lactic acid and does not refer to racemic PDLA(racemic poly-D-lactic acid). Branching can be introduced duringpolymerization of lactic acid, and both “poly-L-lactide” (PLA, PLLA) and“poly-D-lactide” (PDA, PDLA) can refer to branched or unbranchedpolymers of (S)-lactic acid or (R)-lactic acid, respectively. Duringsynthesis of PLA, some (R)-lactic acid can be present, so throughoutthis technology, the PLA can have some impurity, (R)-lactic acid, in thepolymer. Similarly, during synthesis of PDA, some (S)-lactic acid can bepresent, so throughout this technology, the PDA can have some impurity,(S)-lactic acid, in the polymer. Highly pure forms of PLA and PDA aredesirable, but polymerization utilizing chiral monomers can contain somechiral impurity.

As used herein, a “mixture of D- and L-enantiomer polymers” ofpolylactide refers to a mixture of PLA and PDA polymers. For example, amixture can be 95% (weight/weight) PLA and 5% (weight/weight) PDA,wherein the PLA and PDA are separate (non-joined) polymers. Anotherexample of a mixed polymer is a single polymer chain containing 95% PLAand 5% PDA (w/w). Yet another example is a mixture of single polymerchain wherein 95% (weight) of the initial polymer units are PLA and 5%(weight) of the final polymer units are PDA. All mixtures of PLA and PDAof the present technology can contain 0-100% PDA and 0-100% PLA, withthe total of PDA+PLA=100%.

As used herein “2D cell culture” refers to a culture of cells grown on aflat surface. The cells can be grown as a monolayer on Petri dishes,flasks, multi-well plates, or other cell culture containers. Additionalcells optionally can grow over the initial monolayer, forming additionallayers. “3D cell culture” refers to a culture of living cells inside orupon surfaces forming a 3D structure, such as a cell scaffold, which canmimic tissue and organ specific microarchitecture. In 3D cell culture,growth of cells in a 3D arrangement can allow better cell-to-cellcontact and intercellular signaling networks. The 3D cell culture canfacilitate developmental processes allowing cells to differentiate intomore complex structures. 3D cell culture can be described as organotypicculture, involving the growth of cells in a three-dimensional (3-D)environment. A 3D cell culture can be biochemically and physiologicallymore similar to in vivo tissue. In 3D cell culture, cells may becultured and supported in three dimensions, e.g., on and around fibers,beads, sponges, lattices, matrices, or scaffolds.

The term “adherent cells” describes anchorage-dependent cells that arecultured on a suitable substrate that chemically and/or physicallypromotes cell adhesion and spreading. For culturing adherent cells andfor growth of adherent cells, a cell culture container that the cellscan attach to is required. Growth of adherent cells in a cell culturecontainer typically begins with a monolayer of cells attached to oranchored to a growth surface of the cell culture container. The presenttechnology can provide a suitable substrate for culture of adherentcells without any treatment or coating of the culture container. Themajority of cells derived from vertebrates are adherent cells, with theexception of hematopoietic cell lines and certain others. Some adherentcell lines can also be adapted for suspension culture. Many commerciallyavailable insect cell lines grow well in monolayer or suspensionculture.

Anchorage dependence refers to the need for cells to be adhered to or incontact with a solid surface or another layer of cells. Cells can beadhered to other cells or to extracellular matrix, for example. In cellculture containers that require a coating, cells can adhere to a coatingof amino acids or protein affixed to a plastic surface of a conventionalculture container. The coating of amino acids or protein can containcarbonyl groups. Adherent cells can be sensitive to an anchored statethrough physical cytoskeletal signaling, as well as through juxtacrineor gap/tight junction mediated signaling. Typically, epithelial cellsare anchorage dependent and will die if no longer adhered to othercells, a process referred to as anoikis.

The term “biodegradable” refers to polymers that degrade fully (i.e.,down to monomeric species) under long-term composting or otherdecomposition by bacteria or other living organisms, including fungi.Biodegradable polymers are not necessarily hydrolytically degradable andmay require enzymatic action to fully degrade.

As used herein, a “cell culture container” can be a Petri dish, aculture flask, a roller bottle, a microwell or multiwell plate (e.g.,24-well, 96-well, 384-well plates), or any container suitable for cellculture. A “cell culture container” can have one or more 2D growthsurfaces, and/or it can have 3D features for cell growth. The 3Dfeatures, for example, can be inserts, beads, lattices, micro-scaffolds,sponge-like structures, spheroids, web-like structures, and surfaceswithin multiple growth arenas for cell culture.

As used herein, the term “forced flow molding” refers to a method offorming a cell culture container or cell growth structure from apolylactide polymer resin melt at a temperature and pressure that allowthe resin material to readily flow, and the polymer molecules within theresin material to readily become aligned in the direction of resin flowas adequate pressure is applied to the resin material, resulting information of the cell culture container or cell growth structure.

The term “compostable” means that a compound or material is capable ofdisintegrating into natural elements in a compost environment, leavinglittle or no toxicity in the resulting compost material. At least somebeginning of composting typically occurs in about 90 days or less underideal composting conditions. A compost environment can have organicmatter such as dead leaves, twigs, grass clippings, vegetable waste,fruit scraps, coffee grounds, and moisture. The process of compostingcan involve making a heap of wet organic matter and waiting for thematerials to break down into humus after a period of months.

As used herein, the term “electromagnetic radiation” can include radiowaves, microwaves, infrared (IR), visible light, ultraviolet, highenergy electrons or an electron beam, X-rays, and/or gamma rays.

As used herein, the term “enantiomer” refers to a molecule that is amirror image of another; an “enantiomeric pair” is a pair of moleculesthat are mirror images of each other. An enantiomer can contain one ormore chiral centers (stereocenters). A pair of enantiomers differs onlyin the spatial arrangement of their atoms, resulting in enantiomersbeing stereoisomers. Diastereomers are stereoisomers that are not mirrorimages of one another and are non-superimposable on one another.Stereoisomers with two or more chiral centers (stereocenters) can bediastereomers. As used herein, a poly-L-lactide polymer can be anenantiomer of a poly-D-lactide polymer. A poly-L-lactide polymer can bea diastereomer of a poly-D-lactide polymer if, for example, there aredifferences in purity of one or more chiral centers, differences inpolymer chain length or branching, or differences in polymer secondaryform. The polymer PDLLA is a diastereomer of poly-L-lactide and ofpoly-D-lactide.

The “heat deflection temperature” or “heat distortion temperature” (HDT,HDTUL, or DTUL) is the temperature at which a polymer or plastic sampledeforms under a specified load. An example of a method to measure HDT isto expose a plastic to an elevated temperature in air and to observewhether distortion of the plastic occurs.

The term “hydrophilic,” as used herein, refers to the property of havingaffinity for water. Compounds having hydrophilic properties can, forexample, have hydrogen bond accepting functional groups, to which watercan form hydrogen bonds. Hydrophilic polymers (or hydrophilic polymersegments) are polymers (or polymer segments) which can have affinity foraqueous solutions (i.e., are generally water soluble) and/or have atendency to absorb water. In general, the more hydrophilic a polymer is,the more that polymer tends to be wetted by water. The term“hydrophobic,” as used herein, refers to the property of lackingaffinity for, or even repelling water.

For example, the more hydrophobic a polymer (or polymer segment), themore that polymer (or polymer segment) tends to not be wetted by water.

The kilogray is a derived metric (SI) measurement unit of absorbedradiation dose of ionizing radiation, e.g. high energy electrons, X-raysor gamma rays. One kilogray is equal to one thousand gray (1000Gy), andthe gray is defined as the absorption of one joule of ionizing radiationby one kilogram (1 J/kg) of matter.

The term “microparticle” refers to any particle having at least onedimension on the microscale. The term “microscale” refers to a featureor structure having at least one dimension in the range from about 1micron to about 1000 microns. The term “nanoscale” refers to a featureor structure having at least one dimension in the range from about 1 nmto about 999 nm.

The term polymer “molecular weight” can have different meanings. Theterm can refer to “average molecular weight” (Mi) that is the molecularweight as calculated by the weight of the molecule that is mostprevalent in the mix that makes up a molecule. The term canalternatively refer to “number average molecular weight” (Mn), which isthe molecular weight determined by counting the number of tall thedifferent-sized molecules t in a mixture of polymer molecules anddetermining the weight, Mn, for which half the number of molecules inthe mixture are larger and half are smaller than that Mn value. Or, theterm can refer to “weight average molecular weight” (Mw), which is themolecular weight as calculated by taking all the different weightmolecules in a mixture of polymer molecules and determining the weight,Mw, for which half the total weight of molecules is greater and half isless than that Mw value. The units for the molecular weight aretypically Dalton (Da) or kilodalton (KDa, plural kilodaltons).

The dispersity (Ð) or polydispersity index (PDI) is a measure of thedistribution of molecular mass in a given polymer sample. The Ð or PDIof a polymer is calculated as PDI=Mw/Mn, where Mw is the weight averagemolecular weight and Mn is the number average molecular weight. Mw ismore sensitive to polymer molecules of high molecular weight, while Mnis more sensitive to polymer molecules of low molecular weight. The Ð orPDI of a polymer indicates the distribution of individual molecularweights in a collection or batch of polymers. The Ð or PDI has a valueequal to or greater than 1, but as the polymers in a given batchapproach the same chain length, the Ð or PDI approaches 1. A polymermaterial is denoted by the term disperse, or non-uniform, if its chainlengths vary over a wide range of molecular masses.

As used herein, the term “transparent” means allowing visible light topass through, such that shapes and objects can be discerned or focusedupon utilizing the light passing through, either without or with a lightmicroscope, such as to discern cellular morphologies and cellularorganelles. The term “translucent” means allowing light, but not detailsof shapes, to pass through.

As used herein, the term “untreated surface” refers to a surface of acell culture container that has not been exposed to a treatment otherthan sterilization, or coated to improve cell adherence upon thesurface. By comparison, various treatments and coatings are known toimprove cell adherence upon a surface, and the treatments include avariety of physio-chemical processes such as microwave plasma oxygen gastreatment, poly-L-lysine or poly-D-lysine coatings or protein coatingsapplied to polystyrene container surfaces.

As used herein, the Vicat softening temperature (VST) is the temperatureat which a standard indenter (a flat-ended needle with a 1 mm² circularor square cross-section) penetrates 1 mm into the surface of a plastictest specimen under a constant load when the temperature is increased ata uniform rate. For the “Vicat A test”, a load of 10 N is used. For the“Vicat B test”, the load is 50 N. A Vicat softening temperature can beutilized to measure a softening point for materials that have nodefinite melting point, such as polymers, plastics, and glasses.

As used herein, a plastic cell culture container “without surfacemodification” refers to a container whose surfaces substantially retaintheir structure, chemical properties, and physical properties as theyexisted after formation of the plastic container from a resin, and whichhave not been treated other than to a sterilization process as requiredfor cell culture. Surface modification would include, for example,surface melting, abrasion, oxygen and/or nitrogen plasma treatment,chemical addition/modification, or coating addition, such as coatingwith one or more biomolecules such as proteins, peptides, nucleic acids,lipids, proteoglycans, or polysaccharides, or coated with a polymer notpresent in the body of the container. In some surface-modified cellculture containers, oxygen plasma microwave treatment adds oxygen to apolystyrene plastic surface, or protein, or polypeptide coatingsincluding positively charged poly-L-lysine or poly-D-lysine of variousmolecular weights bind to a negatively charged plastic surface such aspolystyrene and contain carbonyl groups or oxygen atoms that can extendfrom a charged plastic surface and interact with cells; such treatmentsor coatings are unnecessary and can be excluded in the presenttechnology, wherein the cell culture container can be used for culturingadherent cells and non-adherent cells without substantial surfacemodification. The surfaces of cell culture containers of the presenttechnology can allow mammalian cell adhesion and growth without the needfor any specific surface modification.

The term “about” as used herein, generally refers to a particularnumeric value that is within an acceptable error range as determined byone of ordinary skill in the art, which will depend in part on how thenumeric value is measured or determined, i.e., the limitations of themeasurement system. For example, “about” can mean a range of ±20%, ±10%,or ±5% of a given numeric value.

As used herein, the terms “highly pure” and “high purity” are defined asa material having a purity of about 95-100%, 96-100%, 97-100%, 98-100%,99-100%, 99.9-100%, 99.99-100%, or 99.999%-100%. As used herein,“chirally pure” means about 95-100%, 96-100%, 97-100%, 98-100%, 99-100%,99.9-100%, 99.99-100%, or 99.999%-100% of the (S) chiral centers inpoly-L-lactide are (S) and of the (R) chiral centers in poly-D-lactideare (R), respectively. As such, chiral purity can be specified as atleast 80%, at least 90%, at least 95%, at least 98%, at least 99%, andat least 99.5%.

The technology is further summarized by the following list of features.

1. A cell culture container comprising a mixture of polylactidepolymers, the mixture comprising (100−X) wt % of poly-L-lactide and X wt% of poly-D-lactide, wherein X≤10, and wherein 100 wt % represents thetotal weight of polylactide polymers in the mixture.2. The cell culture container of 1, wherein the material consists of(100−X) wt % of poly-L-lactide and X wt % of poly-D-lactide, whereinX≤10.3. The cell culture container of any of 1-2, wherein X is from 0.5 to10.4. The cell culture container of 3, wherein X is from 0.5 to 5.5. The cell culture container of any of 14 wherein the poly-L-lactide isat least 99% chirally pure; wherein the poly-D-lactide is at least 99%chirally pure; and wherein the poly-L-lactide and the poly-D-lactide areeach at least 99% unbranched polymers.6. The cell culture container of any of 1-5, wherein the material has aVicat softening temperature greater than 60° C.7. The cell culture container of any of 1-6, wherein the container isbiodegradable.8. The cell culture container of any of 1-7, wherein the container iscompostable.9. The cell culture container of any of 1-8, wherein the material istransparent.10. The cell culture container of any of 1-9 that isviability-sustaining for a live culture of anchorage-dependent cells.11. The cell culture container of 10, wherein >50% of cells in theculture remain ATP positive at 24 hours after cell seeding.12. The cell culture container of any of 1-11 that isviability-sustaining for a live culture of suspension-growing cells,suspension-adapted cells, anchorage-dependent cells, and cells growingon a gelled culture medium.13. The cell culture container of 12, wherein >50% of cells in theculture remain ATP positive at 24 hours after cell seeding.14. The cell culture container of any of 1-13, wherein the container isconfigured as a container selected from the group consisting of a Petridish, a cell culture flask, a multi-well plate, and a roller bottle.15. The cell culture container of any of 1-14, further comprising one ormore anchorage-dependent cells attached to a growth surface in thecontainer.16. The cell culture container of any of 1-15, further comprising one ormore suspension-dependent or suspension-adapted cells within a liquidculture medium in the container.17. The cell culture container of any of 1-16, further comprising one ormore cell growth structures selected from the group consisting ofchannels, lattices, matrices, webs, sponges, fibers, scaffolds, andbeads; wherein said one or more cell growth structures comprise amixture of polylactide polymers, the mixture comprising (100−X) wt % ofpoly-L-lactide and up to X wt % of poly-D-lactide, wherein X≤10, andwherein 100 wt % represents the total weight of polylactide polymers inthe mixture.18. The cell culture container of any of 1-17, wherein a cell growthsurface of the container is devoid of any surface coating or chemical orphysical surface modification.19. A cell growth structure comprising a material comprising (100−X) wt% of poly-L-lactide and X wt % of poly-D-lactide, wherein X≤10.20. The cell growth structure of 19, wherein X is from 0.25 to 10.21. The cell growth structure of 20, wherein X is from 0.5 to 5.22. The cell growth structure of any of 19-21, wherein thepoly-L-lactide is at least 99% chirally pure; wherein the poly-D-lactideis at least 99% chirally pure; and wherein the poly-L-lactide and thepoly-D-lactide are each at least 99% unbranched polymers.23. The cell growth structure of any of 19-22 that is configured as astructure selected from the group consisting of channels, lattices,matrices, webs, sponges, fibers, scaffolds, and beads.24. A method of making a cell culture container, the method comprising:

(a) heating a mixture of polylactide polymers comprising (100−X) wt % ofpoly-L-lactide and X wt % of poly-D-lactide, wherein X≤10 to a moldingtemperature, and wherein 100 wt % represents the total weight ofpolylactide polymers in the mixture;

(b) molding the mixture to form a structure having a shape of said cellculture container;

(c) cooling the structure; and

(d) optionally annealing the structure for an annealing time at anannealing temperature;

whereby said cell culture container is obtained.

25. The method of 24, wherein X is from 0.25 to 10.

26. The method of 25, wherein X is from 0.5 to 5.

27. The method of any of 24-26, wherein the poly-L-lactide is at least99% chirally pure; wherein the poly-D-lactide is at least 99% chirallypure; and wherein the poly-L-lactide and the poly-D-lactide are each atleast 99% unbranched polymers.

28. The method of any of 24-27, wherein annealing is performed, and theannealing time is from 20 seconds to 5 minutes.

29. The method of any of 24-28, wherein annealing is performed, and theannealing temperature is from 70° C. to 95° C.

30. The method of any of 24-29, wherein the molding comprises injectionmolding.

31. The method of any of 24-30, further comprising sterilizing the cellculture container.

32. The method of any of 24-31 that does not comprise applying a surfacecoating or chemically or physically modifying a surface of the containerto enhance cell adhesion to the surface.

33. The method of any of 24-32, wherein the container isviability-sustaining for an anchorage-dependent cell type.

34. The method of any of 24-33, wherein the container isviability-sustaining for suspension-grown cells within a liquid culturemedium in the container.

35. A method of culturing cells, the method comprising:

(a) providing the cell culture container of claim 1;

(b) adding cells and a culture medium to the container; and

(c) incubating the container, cells, and culture medium for anincubation time and at an incubation temperature such that the cells areviably sustained and optionally reproduce.

36. The method of 35, further comprising after step (c):

(d) allowing the container to be biodegraded and/or composted.

37. The method of any of 35-36, wherein the cells comprise ananchorage-dependent cell type.

37a. The method of any of 36-37, wherein the anchorage-dependent celltype adhere to a growth surface of the cell culture container, whereinsaid growth surface is devoid of any surface coating or chemical orphysical surface modification.

38. A cell culture container consisting essentially of a polylactidepolymer material, the material consisting of (100−X) wt % ofpoly-L-lactide and X wt % of poly-D-lactide, wherein X≤10, wherein 100wt % represents the total weight of polylactide polymers in thematerial, and wherein the cell culture container is formed from thepolymer material by a forced flow molding method and has been sterilizedby gamma irradiation or electron beam irradiation, thereby increasingthe hydrophilicity of a cell growth surface of the container compared tothe cell growth surface without said irradiation.39. The cell culture container of feature 38, wherein the forced flowmolding method is selected from the group consisting of blow molding,rotational blow molding, injection blow molding, injection stretch blowmolding, extrusion molding, extrusion molding followed by thermoforming,thin-wall injection molding, micro injection molding, and gas-assistedinjection molding.40. The cell culture container of any of features 38-39, wherein thecontainer is biodegradable.41. The cell culture container of any of features 38-40, wherein thecontainer is compostable.42. The cell culture container of any of features 38-41, wherein thematerial is transparent.43. The cell culture container of any of features 38-42 that isviability-sustaining for a live culture of anchorage-dependent cells.44. The cell culture container of any of features 38-43 that isviability-sustaining for a live culture of suspension-growing cells,suspension-adapted cells, anchorage-dependent cells, and cells growingon a gelled culture medium.45. The cell culture container of any of features 38-44, wherein thecontainer is configured as a container selected from the groupconsisting of a Petri dish, a cell culture flask, a multi-well plate,and a roller bottle.46. The cell culture container of any of features 38-45, including oneor more cell growth structures selected from the group consisting ofchannels, lattices, matrices, webs, sponges, fibers, scaffolds, andbeads; wherein said one or more cell growth structures consistessentially of a polylactide polymer material, the material consistingessentially of (100−X) wt % of poly-L-lactide and up to X wt % ofpoly-D-lactide, wherein X≤10, and wherein 100 wt % represents the totalweight of polylactide polymers in the mixture.47. The cell culture container of any of features 38-46, wherein saidcell growth surface of the container is devoid of any surface coating orchemical or physical surface modification other than by gammairradiation or electron beam irradiation.48. The cell culture container of any of features 38-47, wherein X=0.49. The cell culture container of any of features 38-48, wherein saidirradiation is at a dose in the range from about 25 kGy to about 55 kGy.50. The cell culture container of any of features 38-49, wherein theincrease in hydrophilicity is associated with an increase in tilt angleof a water droplet on said cell growth surface after said gammairradiation or electron beam irradiation.51. A cell growth structure consisting essentially of a polylactidepolymer material consisting of (100−X) wt % of poly-L-lactide and X wt %of poly-D-lactide, wherein X≤10, and wherein the cell growth structureis formed by a forced flow molding method and has been sterilized bygamma irradiation or electron beam irradiation, thereby increasing thehydrophilicity of a cell growth surface of the cell growth structurecompared to the cell growth surface without said irradiation.52. The cell growth structure of feature 51, wherein the forced flowmolding method is selected from the group consisting of blow molding,rotational blow molding, injection blow molding, injection stretch blowmolding, extrusion molding, extrusion followed by thermoforming,thin-wall injection molding, micro injection molding, and gas-assistedinjection molding.53. A method of making a cell culture container, the method comprising:

(a) heating a polylactide polymer material consisting of (100−X) wt % ofpoly-L-lactide and X wt % of poly-D-lactide, wherein X≤10 to a moldingtemperature, and wherein 100 wt % represents the total weight ofpolylactide polymers in the mixture;

(b) subjecting the material to a molding method comprising forced flowmolding to form a structure having a shape of said cell culturecontainer;

(c) cooling the structure;

(d) optionally annealing the structure for an annealing time at anannealing temperature; and

(e) sterilizing the container obtained from step (c) or step (d) withgamma irradiation or electron beam irradiation;

whereby said cell culture container is obtained.

54. The method of feature 53, wherein the forced flow molding method isselected from the group consisting of blow molding, rotational blowmolding, injection blow molding, injection stretch blow molding,extrusion molding, extrusion followed by thermoforming, thin-wallinjection molding, micro injection molding, and gas-assisted injectionmolding.55. A method of culturing cells, the method comprising:

(a) providing the cell culture container of feature 38;

(b) adding cells and a culture medium to the container; and

(c) incubating the container, cells, and culture medium for anincubation time and at an incubation temperature such that the cells areviably sustained and optionally reproduce.

56. The method of feature 55, further comprising after step (c):

(d) allowing the container to be biodegraded and/or composted.

57. The method of feature 56, wherein the cells comprise ananchorage-dependent cell type, and wherein the anchorage-dependent celltype adheres to said growth surface of the cell culture container,wherein said growth surface is devoid of any surface coating or chemicalor physical surface modification other than by gamma irradiation orelectron beam irradiation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic representation of an embodiment of a method ofmaking a transparent cell culture container with 2D and/or 3D growthsurfaces.

DETAILED DESCRIPTION

The present technology provides cell culture containers made from blendsof poly-L-lactide (PLA) and poly-D-lactide (PDA) which blends aretransparent, have good temperature and mechanical stability, and arebiodegradable and compostable. These cell culture containers also canprovide excellent anchorage for adherent cells without any celladhesion-promoting coating or surface treatment.

PLA is a readily biodegradable polymer which has been used to produce avariety of plastic consumer articles, typically by injection molding attemperatures in the range from about 178° C. to about 240° C. PLA has amelting temperature in the range from about 157° C. to about 170° C.However, molded products made from PLA have a heat deflectiontemperature from about 49° C. to about 52° C., and their Vicat softeningtemperature is about 50° C. This makes pure PLA unsuitable for makingcell culture containers, as the containers would deform at temperaturesgreater than about 50° C. The present inventor has discovered thatsuitable cell culture containers with highly desirable properties ofheat stability, transparency, biodegradability, and compatibility withadherent cells can be made by combining PLA with PDA to form a polymerblend before molding.

Enantiomers generally have similar chemical properties, except when theyinteract with other chiral compounds or with some enzymes. Thus, themelting temperature, heat deflection temperature, and Vicat softeningtemperature of a sample of PDA will be the same as a sample of PLA iftheir polymer MW and branching are the same. Surprisingly, by mixingcertain proportions of PDA with PLA, it is possible to form cell culturecontainers with a high tolerance for heat. The cell culture containercan be transparent and can be compostable. Unexpectedly, such cellculture containers can be used with adherent cells without the need forchemical or physical treatments or applying adhesion-promoting coatings.

PLA and PDA polymers for use in the present technology can be of anymolecular weight useful to produce suitable cell culture containers. Forexample, the molecular weight can be a value specified as suitable bythe resin manufacturer for manufacturing containers by injectionmolding, blow-molding, or thermoforming. For an injection-molding resin,a typical polylactide molecular weight can be about 100,000 Da or fromabout 75,000 to 125,000 Da, or from about 50,000 to 150,000 Da. forexample. The polymers have any dispersity (Ð) and any level of polymerbranching consistent with the desired properties of suitability formanufacturing and use as a cell culture container, or desired values ofVicat softening temperature, transparency, biodegradability, and/or cellattachment.

The PLA and PDA polymers utilized to form cell culture containers hereincan have various substituents on various methyl groups or variouscarbonyl groups. For example, Formula 5 illustrates substituents R1, R2,and R3 on methyl groups in PLA, PDA, or a diastereomer of PLA or PDA. Ingeneral, the value of n can be at least 100, 200, 300, 500, 1000, 1400,1500, 2000, 3000, 4000, or 5000 or more. Optionally coupled with any ofthese lower limits, as appropriate, the value of n can be less than orequal to 10000, 5000, 3000, 2000, 1500, 1200, 1000, or 500.

Formula 6 illustrates substituents R4, R5, and R6 on the carbonyloxygens of PLA, PDA, or a diastereomer of PLA or PDA. In general, thevalue of n can be at least 100, 200, 300, 500, 1000, 1400, 1500, 2000,3000, 4000, or 5000 or more. Optionally coupled with any of these lowerlimits, as appropriate, the value of n can be less than or equal to10000, 5000, 3000, 2000, 1500, 1200, 1000, or 500.

The substituents R1, R2, R3, R4, R5, and R6 (R1-R6) in Formulas 5 and 6can be independently selected and can all be the same or different.Substituents on methyl groups, for example in Formula 5, can be present(or combined) with substituents on carbonyl groups, for example inFormula 6. The substituents R1, R2, and R3 (R1-R3) can be present butwithout any substituents R4, R5, and R6 (R4-R6) on carbonyls. R1-R3 canbe hydrogen and R4-R6 can be no substituents. For example, R1-R3 can behydrogen, hydroxy, sulfoxy, halo, acyl, acyloxy, alkyl, heteroalkyl,alkenyl, heteroalkenyl, alkynyl, heteroalkynyl, alkoxy, cycloalkyl,heterocycloalkyl, aryl, arylalkyl, arylhalo, arylhydroxy, arylcyano,aryltrifluoromethyl, aryltrifluoromethoxy, arylnitro,aryltrifluoro-methoxy, aryl ether, aryl ester, aryl sulfonyl, arylsulfinyl, aryl sulfonamidyl, aryl sulfonate, aryl sulfoxyl, arylphosphate ester, aryl carbonyl, aryl carboxylate, aryl carbamate, arylamine, aryl imide, heteroaryl, heteroarylalkyl, heteroarylhalo,heteroarylhydroxy, heteroarylcyano, hetero-aryltrifluoromethyl,aryltrifluoromethoxy, arylnitro, heteroaryltrifluoromethoxy,heteroarylnitro, heteroarylether, heteroarylester, heteroarylsulfonyl,heteroarylsulfinyl, heteroarylsulfonamidyl, heteroarylsulfonate,heteroarylsulfoxyl, heteroarylphosphate ester, heteroarylcarbonyl,heteroarylcarboxylate, heteroarylcarbamate, heteroarylamine,heteroarylimide, quinidine, morpholine; any ring structure can beoptionally substituted with any of the substituents described herein;and any two adjacent substituents can come together to form acarbocyclic or heterocyclic ring system. The substituents R4-R6 caninclude any the aforementioned substituents for R1-R3 that are suitablefor covalent attachment to oxygen.

The PLA or PDA polymers utilized in the present technology can beneutral or can have positive or negative charges on various substituents(e.g., protonated tertiary amino groups or deprotonated carboxylgroups). The PLA and PDA can have negative charge on one or more oxygenatoms as is shown in Formula 7, where associated cations A+, B+, and D+can be the same or different. A+, B+, and D+ can have multivalent chargeand can form salts with the polylactide polymer chain in hemi, mono,bis, and other configurations. The negative charge on an oxygenillustrated in Formula 7 can be a partial charge, as resonant structuresare possible with a carbonyl adjacent to O—, further illustrating thepossibility for hemi, mono, bis, and other salt configurations.

A+, B+, and D+ can be independently organic or inorganic cations, atrivalent metal cation, or a monovalent alkali earth metal cation. InFormula 7, the D+ cation can be absent and the negatively charged oxygenassociated with D+ can be a carbonyl, such that A+ and B+ form saltswithout presence of D+. Although not illustrated in Formula 7, organicanions, for example a borate, can interact with various functionalgroups either covalently attached to the polylactides or to cations inionic attachment to the polylactides.

The PLA and the PDA utilized to form cell culture containers can havevarious, independently selected, functional groups on the terminal endsof the PLA and the PDA. Functional groups on the terminal ends arevarious functional groups that can be covalently attached to theterminal ends of a main polymer backbone (chain) of PLA or PDA and toterminal ends of branching polymer chains extending from the mainpolymer backbone, or from other branching polymer chains. Formula 8represents a non-limiting example of an amine functional group attachedto a terminal end of PLA.

The amine functional group illustrated in Formula 8 can represent anyother suitable functional group for attachment to terminal ends of PLAor PDA. Non-limiting examples of functional groups are esters, thiols,propargyl (from alkyne propyne), azide, acrylate, methacrylate, orN-2-hydroxyethylmaleimide. The functional groups attached to terminalends can form bridges between or among polymer chains.

The PLA and the PDA utilized to form cell culture containers can haveindependently selected, joining functional groups on the terminal endsof the PLA and the PDA. Joining functional groups can attach to one ormore terminal ends of PLA or PDA and join the PLA or PDA to otherpolymers of PLA or PDA. In this way, one or more joining functionalgroups can extend the polymer length of PLA or PDA. Any joining groupsuitable for attachment to PLA or PDA, with further attachment to PLA orPDA can be utilized. A non-limiting example is a secondary amine that isattached to a PLA polymer on both ends. For example, the primary amineillustrated in Formula 8 can be a secondary amine with PLA polymersattached on both sides of the secondary amine.

Addition of compounds, salts, or chemicals to PLA can increase the Vicatsoftening temperature and the heat deflection temperature, but canresult in a loss of transparency. For example, the addition of talc toPLA can be utilized to produce opaque cutlery and dishware that can havetolerance for temperatures over 50° C. The present cell culturecontainers can contain additional materials other than PLA and PDA. Ifmaterials other than PLA and PDA are added, materials and/or theiramounts preferably are selected such that the transparency of theresulting cell culture containers is maintained. For example, PEG(polyethylene glycol), PPG (polypropylene glycol), or other plasticizerscan be added. Releasing agents, coloring agents, and/or crystallizationagents can be added. Optionally, compounds can be added to improvetransparency or to filter or block total transmission of ranges of theelectromagnetic spectrum, such as to provide color or to protect cellswithin the container from certain wavelengths of electromagneticradiation. Optionally, micro or nanoparticles can be added, for example,to block or partially filter UV light.

Surface coatings such as poly-D-lysine, collagen, antibodies, oraptamers can optionally be added to the cell culture containers toenhance cell adhesion or selectively adhere certain desired cell types.For example, addition of carbonyl groups or addition of oxygen atoms toa growth surface of a cell culture container can improve cell adhesion,and the oxygen atoms can act as hydrogen bond acceptors. While notintending to limit the technology to any particular mechanism, themixtures of PLA and PDA disclosed herein can be used to form cellculture containers can produce surfaces having exposed carbonyl groupsor oxygen atoms without the need for any treatment or coating step. Theexposed carbonyl groups or oxygen optionally can be combined with otherfunctional groups. Such exposed carbonyl groups or oxygen atoms canincrease the hydrophilicity of a surface.

The present technology provides a method to produce cell adhesive,compostable (biodegradable), and transparent, cell culture containers.The method can comprise mixing PLA with a selected percentage(weight/weight) of PDA. The percentage of PDA can be, for example, about0.5% to 50%, 1% to 50%, about 2% to 50%, about 2% to 30%, about 2% to20%, about 2% to 10%, about 2% to 5%, about 1% to 20%, about 1% to 10%,about 1% to 5%, about 3% to 20%, about 3% to 10% about 4% to 20%, about4% to 10%, about 5% to 20%, or about 5% to 10% by weight. Differentmixtures or ranges of PDA content can be chosen depending on theintended use of the containers. For example, while 50% PDA can yield ahigher Vicat softening temperature than a lower percentage, the use of50% PDA also can increase the cost of a cell culture container. Asanother example, 1.0% PDA can enable a cell culture container towithstand temperatures of 70° C. water (see Example 1), but for someapplications 5% PDA can be used so as to provide still higher thermalstability (>70° C.). Blends containing as little as 0.5% PDA, if usedwith annealing, can yield a cell culture container capable ofwithstanding 70° C. water.

The Mn or “number average molecular weight” of the PLA and the PDAutilized herein can be greater than about 1 KDa, greater than about 5KDa, greater than about 10 KDa, greater than about 20 KDa, greater thanabout 30 KDa, greater than about 40 KDa, greater than about 50 KDa,greater than about 60 KDa, greater than about 70 KDa, greater than about80 KDa, greater than about 90 KDa, greater than about 100 KDa, greaterthan about 200 KDa, greater than about 300 KDa, greater than about 400KDa, and greater than about 500 KDa.

The Mw or “weight average molecular weight” of the PLA and the PDAutilized herein can be greater than about 2 KDa, greater than about 10KDa, greater than about 20 KDa, greater than about 40 KDa, greater thanabout 60 KDa, greater than about 80 KDa, greater than about 100 KDa,greater than about 120 KDa, greater than about 140 KDa, greater thanabout 160 KDa, greater than about 180 KDa, greater than about 200 KDa,greater than about 300 KDa, greater than about 400 KDa, greater thanabout 500 KDa, greater than about 600 KDa, greater than about 700 KDa,greater than about 800 KDa, greater than about 900 KDa, and greater thanabout 1000 KDa.

The PLA and the PDA utilized herein can have high chiral purity suchthat greater than 95% of the (S) chiral centers are (S) or greater than95% of the (R) chiral centers are (R), greater than 98% of the (S)chiral centers are (S) or greater than 98% of the (R) chiral centers are(R), and greater than 99% of the (S) chiral centers are (S) or greaterthan 99% of the (R) chiral centers are (R), respectively.

A method to produce a cell adhesive, compostable (biodegradable), andtransparent, cell culture container can consist essentially of mixingPLA (100−X) % with a percentage (weight/weight) of PDA (X %). While thePLA and PDA are essential, other ingredients can be added that do notcause lack of transparency in a selected range from between about 380 toabout 760 nm. Other ingredients can be added that do not make themixture non-biodegradable. Other ingredients can be added that do notmake the final cell culture container non-adherent to adherent cells. Toconsist essentially of can mean other ingredients (besides PLA and PDA)added do not cause lack of transparency, lack of biodegradability, orlack of cell adhesion in a cell culture container. To consistessentially of can mean that a suitable cell culture container isproduced with viable adhesion for adhesive cells. A method to produce acell adhesive, compostable (biodegradable), and transparent, cellculture container can consist of mixing PLA (100−X) % with a percentage(weight/weight) of PDA (X %). The percentage of PDA mixed with PLA canbe selected from the range between 0.1% and 50% (weight/weight).

An example of a method to produce a cell culture container is shown inFIG. 1 . The mixing of PLA and PDA, shown as 101, can be performedutilizing a mixture comprising PLA at (100−X) % (weight/weight) and PDAat X % (weight/weight), where 100% corresponds to the total amount byweight of PLA+PDA in the mixture. The percent of X can be from 0.1% to50%. The mixing in 101 can be performed by any means known in the art,for example, tumbling, wet milling, dry milling, homogenization, sonic(or acoustic, resonant) mixing, and suspension mixing. The mixing in 101can further comprise a drying step or any step to complete the mixing. Adrying step can be to reduce moisture in the mixture before attempting amelting. A mixing step can be followed by, or can include, one or moredrying steps for any purpose. A drying step is not limited intemperature or in vacuum conditions. For example, a drying step canemploy a temperature from about 50° C. to 100° C., about 60° C. to 90°C., or about 70° C. to 80° C.

The mixture can then be heated to a molding temperature in 102 using anymeans known in the art. The molding temperature is not limited. Forexample, a molding temperature can be selected from about 100° C. to600° C., about 125° C. to 300° C., about 150° C. to 275° C., or about175° C. to 250° C.

After heating to a molding temperature, any of several types of forcedflow molding methods, including injection molding, blow molding,rotational blow molding, injection blow molding, injection stretch blowmolding, extrusion molding, and extrusion followed by thermoforming, canbe utilized to shape a cell culture container. Injection molding caninclude high pressure injection of a mixture at or near a moldingtemperature. High pressure injection of a mixture of PLA and PDA, or PLAalone, can be utilized to form intricate parts or dimensions of largercomponents at or near the micrometer or nanometer scale. Other forms ofinjection molding that can be used include thin-wall injection molding,micro injection molding, gas-assisted injection molding, 3D printing canbe utilized to mold, shape, or deposit a mixture of PLA and PDA intointricate parts or dimensions, and is particularly useful for making asmall number of containers having a complex structure. Other types ofmolding such as blow molding, rotational blow molding, injection blowmolding, injection stretch blow molding, extrusion molding, andextrusion followed by thermoforming can be used, and it is preferredthat such methods involve introduction of resin under pressure, and suchtreatment is expected to align the polymer chains and result in densepacking of the chains, which is associated with an advantageous increasein hydrophilicity upon irradiation by gamma irradiation or electron beamirradiation.

Injection molding, or any suitable type of formation or shaping asdescribed above, can occur at 103 wherein a shape of a cell culturecontainer is formed from the mixture 101 at a molding temperature (102).The shape of the cell culture containers disclosed herein is not limitedin any way, and the shape 105 can be a Petri dish, a culture flask, aroller bottle, a microwell or multiwell plate (e.g., 24-well, 96-well,384-well plates), and any dish or container suitable for cell culture.3D features can be separately molded; the 3D features, for example, canbe inserts, beads (106), lattices, micro-scaffolds, sponge-likestructures, spheroids, web-like structures, and surfaces with multiplegrowth arenas for cell culture. After the formation or shaping a coolingstep can be performed at 103 to cool the shaped mixture such that themolded shape is retained.

Cooling can be to any temperature suitable to stabilize a shape of thecontainer or a component thereof.

After injection molding, 3D printing, formation, or shaping at 103, anannealing step can be performed at 104. Annealing can improve thethermal stability of a cell culture container and can impart acrystallinity into the material which can improve thermal stability.Annealing generally refers to exposing the molded part to an selectedtemperature for a selected period of time, and can be carried out by anyknown process, such as tunnel annealing, oven annealing, heat lamp(infra-red) annealing, microwave annealing, radiation annealing, hotwater bath annealing, hot air annealing, vacuum annealing, or sonicannealing. The annealing time can be about 1 to 15 minutes at atemperature in the range from about 20-75° C., or can be about 20seconds or more, such as 20-60 seconds, at 90° C. Annealing time andtemperature can be varied depending on, for example, the percentage ofPDA in mixture 101 (higher levels of PDA enhance the annealing process),the desired Vicat softening temperature, the heat deflection temperatureof products 105, 106, the thickness or shape of the product, or thedesired optical clarity of the product (excessive annealing reducesoptical clarity). Annealing time and temperature also can be varied dueto humidity conditions, properties of the mixture, additives other thanPLA and PDA in the mixture, and desired crystal forms. Further,annealing time and temperature can be varied based upon the molecularweights of the PLA and the PDA polymers. If a sufficiently highannealing temperature is used, the time of annealing can be shortenedsuch that annealing takes place within the mold, and no separateannealing process is required. See, for example, Product Data Sheet forLUMINY D070 from Total Corbion PLA BV, Revision 7 May 2019, which ishereby incorporated by reference. It is within the routine skill in theart of plastics molding to routinely determine optimum annealingconditions for each molded part and polymer resin. In the presenttechnology, the following parameters offer the best control over theannealing conditions: amount of PDA in the PLA/PDA mix, annealingtemperature, and annealing time. The annealing conditions should beadjusted so as to obtain the desired heat stability of the part (e.g.,as measured by Vicat softening temperature or other parameters) andoptical clarity (for compatibility with light microscopy). Withoutintending to limit the technology to any particular mechanism, theannealing conditions are believed to impact the crystalline state of thepolymers, which determine the heat stability and optical properties.

A lid 107 can optionally be made from mixture 101. The lid 107 shown inFIG. 1 can be formed by 101, 102, 103, 104, for example. Lid 107 andcontainer 105 can comprise, for example, inserts, extensions, rods,lattices, dividers, well, observation areas, and notches, threads, orgrooves for fastening lid 107 to container 105. The container 105, the3D spheroids or beads 106, and lid 107 shown in FIG. 1 can form a unit,which can be stackable, which can interlock with adjacent units, orwhich can be combined with other units to form a larger 3D cell culturecontainer. Optionally, container 105 can form a unit alone. Lid 107 canbe formed from an entirely different material than the body of the cellculture container, particularly if no cell growth is desired on the lid.

Annealing can increase or cause a crystallization of the mixturecomprising PLA and PDA. The crystallization can increase the heattolerance of a cell culture container, for example, increasing the Vicatsoftening temperature or the heat deflection temperature. Annealing canbe done at a time or a temperature that does not destroy thetransparency of a cell culture container. For example, transparency canbe halted when one or more crystal forms initiate change to a differentcrystal form. Annealing can cause a glass transition. Optionally,annealing can cause a transition to an amorphous form. Annealing can beperformed in the injection mold or in the shaping of the cell culturecontainer. Annealing can be performed after sterilization.

The cell culture containers disclosed herein can have a Vicat softeningtemperature or a heat deflection temperature greater than about 50° C.,greater than about 60° C., greater than about 70° C., greater than about80° C., greater than about 90° C., greater than about 100° C., greaterthan about 110° C., greater than about 120° C., and greater than about130° C. Thermal stability of the cell culture containers disclosedherein can be measured by incubating the cell culture containers in airfor 30 minutes at a testing temperature and by observing whether anyvisible warping of a flat bottom of the cell culture containers occurs.Thermal stability of the cell culture containers disclosed herein can begreater than about 60° C., greater than about 65° C., greater than about70° C., greater than about 75° C., greater than about 80° C., greaterthan about 85° C., greater than about 90° C., greater than about 95° C.,greater than about 100° C., greater than about 110° C., greater thanabout 120° C., and greater than about 130° C.

A growth surface area of a cell culture container can be larger thanabout 1 mm², larger than about 1 cm², larger than about 5 cm², thanabout 50 cm², larger than about 100 cm², larger than about 1 m², largerthan about 5 m², or larger than about 10 m². The cell culture containersdisclosed herein can have 3D features causing a large growth surfacearea to volume ratio in the cell culture containers. Growth surface areato volume can be measured by dividing growth surface area by volume. Forexample, 8 cm² divided by 2 cm³=4 cm⁻¹. The growth surface area tovolume ratio of the cell culture containers disclosed herein can haveany desired value, or can be at least about 0.1 cm⁻¹, at least about 0.5cm⁻¹, at least about 1 cm⁻¹, at least about 5 cm⁻¹, at least about 10cm⁻¹, at least about 25 cm⁻¹, at least about 50 cm⁻¹, or at least about100 cm⁻¹.

The cell culture containers disclosed herein preferably can contain hotwater or hot aqueous solutions without deforming. For example, hot watercan be poured into the cell culture containers without deformation ofthe cell culture containers. Optionally, the cell culture containersdisclosed herein can withstand, without deformation, hot water at atemperature greater than about 70° C., greater than about 75° C.,greater than about 80° C., greater than about 85° C., greater than about90° C., greater than about 95° C., or greater than about 100° C.Optionally, agar can be gelled in a cell culture container disclosedherein without deforming the container.

The cell culture containers disclosed herein can be transparent.Transparency can be measured by total transmittance. For example, totaltransmittance can equal incident light minus (absorption+reflection).Optionally, the total transmittance of the cell culture containersdisclosed herein can be greater than 10%, greater than 20%, greater than30%, greater than 40%, greater than 50%, greater than 60%, greater than70%, greater than 80%, or greater than 90%. Transparency can indicatelack of components or crystal forms with different indices ofrefraction. Transparency can be measured in one or more ranges or in oneor narrow bands in the visible light spectrum from about 380 or 400 nmto about 700, 740, or 760 nanometers. Certain ranges between about 380to about 760 nanometers can be made non-transparent for specific cellcultures, for example, by coatings, additives, or treatments (e.g.,annealing). Coatings, additives, or treatments (e.g., annealing) can beapplied to block or to inhibit electromagnetic waves in any desiredrange of the spectrum of electromagnetic radiation. If only one or moreselected wavelength ranges are blocked, the cell culture container isstill generally considered to be transparent. Particulates can be addedto the mixture of PLA and PDA during mixing or after mixing. Theparticulates can be utilized, for example, for heat (IR) or UVabsorption, and the particles may lower total transmittance whileallowing transparency in one or more ranges between 380 to about 760 nm.

The cell culture containers can be sterilized after annealing.Optionally, sterilization can be accomplished by annealing or by asterile manufacturing technique. Sterilization can be performed by anymeans known in the art, such as gamma irradiation or irradiation by aparticle beam or electron beam.

The cell culture containers disclosed herein can be manufactured bymixing about 0.1% to about 50% PDA with PLA, melting the mixture to asuitable molding temperature, molding the melted mixture, cooling to asuitable release temperature, and then annealing the cell culturecontainer. If pure PLA and PDA are utilized, the cell culture containerscan comprise carbonyl groups extending from the PLA and PDA on the outersurfaces. The carbonyl groups can act as hydrogen bond acceptors. Thecell culture containers can retain a water droplet, about 100microliters in volume, without movement of the water droplet, at anangle in non-limiting examples, from about 0°-75°, about 0°-65°, about0°-45°, about 0°-35°, about 0°-25°, about 0°-20°, about 0°-17°, about0°-15°, and about 0°-12°. Electron beam irradiation (e.g., 25 kGy, 45kGy, 55 kGy) can increase the hydrophilicity.

The cell culture containers disclosed herein can be utilized for cultureof either suspension or suspension-adapted cell types or foranchorage-dependent/adherent cell types, eukaryotic cells, andprokaryotic cells. In the cell culture containers disclosed herein,bacterial E. Coli can be grown in any suitable medium. Luria Brothbacterial growth medium (1% tryptone, 1% NaCl, and 0.5% yeast extract inwater) with 2% dissolved agar is a non-limiting example of a suitablemedium. Yeast cells can be grown in the cell culture containersdisclosed herein.

Surprisingly, the technology disclosed herein can provide cell culturecontainers for adherent cells without surface treatment or coating. Thecell culture containers provided herein also can be used for cells thatgrow in suspension, or that grow both in suspension and attached to asurface or to other cells. The containers can be used for culturingcells derived from multicellular eukaryotes, animal cells, such asmammalian cells, including human cells, plant tissue or cell culture,fungal culture, and microbiological culture. Viral culture can beaccomplished, for example, in the eukaryotic cells grown in thecontainers. For growth upon untreated and uncoated surfaces, anysuitable growth or culture medium can be used. An example of a growthmedium for eukaryotic cell growth is DMEM medium (Dulbecco's ModifiedEagles Medium) supplemented with 10% fetal bovine serum, 2 mML-glutamine, and 100 I.U./ml each of penicillin and streptomycin. Thecontainer can be placed in a 37° C. incubator with an atmosphere of 5%CO₂ in air. Because the growth surfaces of the containers providedherein do not necessitate treatment or coating before cultivation orgrowth, large surface area growth surfaces, 3D growth surfaces, andintricate growth surfaces can readily be produced more conveniently andat less cost than containers requiring surface treatment or coating.

Cell culture containers according to the present technology can sustainthe viability of cells cultured therein. Both suspension-growing orsuspension-adapted and anchorage-dependent cells can be viably sustainedby containers of the present technology, as well as cells such asbacteria, yeast, or other fungi which grow on gelled culture medium. Theviability sustaining feature of the containers can be assayed by any ofa variety of known methods. For example, seepromega.com/resources/guides/cell-biology/cell-viability/. Cellviability can vary depending on the cell species, seeding density, ageof the cell culture following seeding, and other factors.

Cell culture containers of the present technology can be assessed, forexample, by determining the fraction of cells that are measured as ATPpositive. Cell culture containers of the present technology can beviability sustaining, for example, in that they maintain greater than50%, or greater than 60%, or greater than 70%, or greater than 80%, orgreater than 90% of cells in the culture as ATP positive at 24 hoursafter seeding the culture.

The technology described herein can provide cell culture containers ofany size or shape for cell cultivation. The transparent cell culturecontainers disclosed herein and the methods disclosed herein can beutilized for cultivation, growth, sustenance, or multiplication ofbacteria, Archaea (e.g, prokaryotes), Protozoa, Chromista (e.g., algae),plants, fungi, animal cells, viruses, or prions. The cell culturecontainers can be utilized for replication of DNA, RNA, amyloids,oligonucleotides, and polypeptides. For detaching adherent cells, thecell culture containers can be used with any detachment method known inthe art, for example, by introducing trypsin into the container. Becausethe cell culture containers of the present technology are highlyversatile, in that they can be utilized to culture a wide variety ofdifferent cell types, including anchorage-dependent cells, cells growingin suspension, cells adapted to growing in suspension, and cells growingon gelled media, the use of these cell culture containers can reduce thesize, complexity, and cost of inventory for a research or cellproduction facility.

The technology described herein can be produced using 3D printing. Forexample, a mixture comprising poly-L-lactide at (100−X) %(weight/weight) and poly-D-lactide at X % (weight/weight), wherein X %is 0.1% to 50%, is introduced into a 3D printer. The heating element ofthe 3D printer can heat the mixture to a molding temperature. Therobotic or moving printing head of the 3D printer can mold the mixturein the shape of a cell culture container (or almost any shape). Theshaped or formed mixture can be cooled. The shaped or formed mixture canbe annealed to form a transparent cell culture container, a 3D structuresuch as a cell scaffold for adherent cell culture and growth.

EXAMPLES Example 1: Manufacture of Petri Dishes

Poly-D-lactide (PDA) was mixed with poly-L-lactide (PLA) in variouspercentages, molded, annealed, and tested to determine the ability tofill a Petri dish with water up to temperatures of 70° C. with novisible deformation in the product. Petri dishes were molded by EcNowTech Inc. (34080 ExCor Road, Albany, Oreg. 97321). Four batches weremade by combining PDA and PLA by weight percentages. Batch 1 was acontrol batch made of 100% PLA. Batch 2 was made up of 99.5% PLA and0.5% PDA. Batch 3 was made up of 99.0% PLA and 1.0% PDA. Batch 4 wasmade up of 95% PLA and 5% PDA.

Materials were mixed in a tumble mixer for approximately ten minutes perbatch. 21 Kg batches were used for each mixture. Each batch was dried at170° F. (76.7° C.) for 6 hours per batch. Each batch was processed on an85-ton Engel injection molding machine using a single cavity Petri dishtool. A hot tip was utilized for direct injection of plastic into thetool. The material was processed after drying at a melt temperature of400° F. (204.4° C.). Post molding, the products were annealed by passingthrough a heat tunnel (oven) at 150° F. (65.6° C.) for 5, 10, or 15minutes to impart crystallinity into the material. It was found that a 5to 10-minute dwell time in the oven was sufficient to impart sufficientcrystallinity without negatively impacting the transparency or clarityof the material. Testing of batches with hot water (70° C.) wasconducted. The testing proved that 0.5% (w/w) of PDA in Batch 2 wassufficient with proper annealing (5-10 minutes) to enable the product towithstand temperatures of 70° C. when water at that temperature waspoured into the Petri dishes.

Injection-molded PLA cell culture containers are typically transparentwith a slightly yellowish cast that does not compromise their use.However, for esthetic purposes the yellowish cast can be corrected byadding a very small amount of optical brightener. Accordingly, one suchoptical brightener chemical, a bis(benzoxazol)stilbene compound wascommercially obtained from the Sukano Polymers Corp. (Duncan, S.C.)already pre-diluted and dispersed in PLA (mixture known as PLA obS515-N). This product was further diluted approximately 100-fold intothe PLA-PDA resin blends and used to prepare injection-molded cellculture containers. Accordingly, the final concentration of opticalbrightener compound in the cell culture container molded parts wastypically about 5-20 ppm, and substantially less than 100 ppm.

Example 2: Water Droplet Movement on 99% PLA with 1% PDA Petri Dishes

Petri dishes were molded by EcNow Tech Inc. by blending about 1%poly-D-lactide polymer with 99% poly-L-lactide polymer (weight/weight)as described in Example 1. Before testing, each Petri dish was washedwith distilled water. An X marking was placed on the outside of eachdish near its center. The X marking was used to monitor initiation ofmovement of a water droplet placed above that marking after tilting eachPetri dish at increasingly steep angles.

Droplets of increasing micropipette-measured volumes (25, 50, and 100microliters) of double-distilled water were applied to the bottom insidesurface of each Petri dish, with the X marking on the outside of thePetri dish. Three droplets of each volume were tested on each Petridish. Two non-irradiated Petri dishes were tested, and two Petri disheswere tested for each irradiated condition. The tilt angle for a Petridish was set and measured using a protractor goniometer. Tables 2-4summarize results for irradiated Petri dishes. Table 1 summarizesresults for non-irradiated Petri dishes, and the measured elevationangle (using the protractor goniometer) is shown in degrees for eachPetri dish and each droplet.

TABLE 1 Droplet Results for Two Non-Irradiated Petri Dishes. Water Dish1, Dish 2, Droplet Size Droplets 1, 2, 3 Droplets 1, 2, 3 (microLiters):(Degrees) (Degrees) 25 17.5, 17, 17 18.5, 18, 17.5 50 11, 11, 10.5 10,11, 10.5 100  7.5, 8, 7.5 6, 6.5, 6

TABLE 2 Droplet Results for Two 25 kGy Electron Beam Irradiated PetriDishes. Water Dish 1, Dish 2, Droplet Size Droplets 1, 2, 3 Droplets 1,2, 3 (microLiters): (Degrees) (Degrees) 25 26, 27.5, 27 28, 28.5, 27 5013, 14, 13 16, 15, 16 100  8, 8.5, 8 11, 11.5, 11.5

TABLE 3 Droplet Results for Two 45 kGy Electron Beam Irradiated PetriDishes. Water Dish 1, Dish 2, Droplet Size Droplets 1, 2, 3 Droplets 1,2 ,3 (microLiters): (Degrees) (Degrees) 25 32, 32.5, 32 30, 31, 31 5017, 18, 18 17.5, 17, 18 100  12, 12.5, 12.5 10.5, 11, 11.5

TABLE 4 Droplet Results for Two 55 kGy Electron Beam Irradiated PetriDishes. Water Dish 1, Dish 2, Droplet Size Droplets 1, 2, 3 Droplets 1,2, 3 (microLiters): (Degrees) (Degrees) 25 28, 29 30 28, 27, 27 50 16,17, 16.5 16.5, 16.5, 17 100  11, 12, 11 12, 11, 11.5As water droplet mass was increased 4-fold (from 25 microLiters to 100microLiters) the approximate elevation angle needed to initiate dropletmovement decreased about 3-fold. Water droplet movement on Petri dishsurface was initiated most easily (with the smallest angle of elevation)with the Petri dishes that were not irradiated. These non-irradiateddishes are therefore considered most hydrophobic. With electron beamirradiation, the most decrease in Petri dish hydrophobicity (e.g.,increased water wettability and increased elevation angle to initiatewater droplet movement on the Petri dish surface) was obtained with anelectron dosage of 25 kGy with very little additional change above thatelectron dosage.

Example 3: Water Droplet Movement on 99% PLA with 1.0% PDA Petri Dishes

Petri dishes were molded by EcNow Tech Inc. by blending about 1%poly-D-lactide polymer with 99% poly-L-lactide polymer (weight/weight)as described in Examples 1 and 2, but these dishes were molded usingdifferent batches of polylactide resins. Before testing, each Petri dishwas washed with distilled water. An X marking was placed on the outsideof each dish near its center. The X marking was used to monitorinitiation of movement of a water droplet placed above that markingafter tilting each Petri dish at increasingly steep angles. Droplets ofincreasing micropipette-measured volumes (25, 50, and 100 microliters)of double-distilled water were applied to the bottom inside surface ofeach Petri dish, with the X marking on the outside of the Petri dish.Three droplets of each volume were tested on each Petri dish. Fournon-irradiated Petri dishes were tested, two Petri dishes were testedfor the 25 kGy irradiated condition, two Petri dishes were tested forthe 40 kGy irradiated condition, and three Petri dishes were tested forthe 55 kGy irradiated condition. The tilt angle for a Petri dish was setand measured using a protractor goniometer. Tables 6-8 summarize resultsfor irradiated Petri dishes. Table 5 summarizes results fornon-irradiated Petri dishes, and the measured elevation angle (using theprotractor goniometer) is shown in degrees for each Petri dish and eachdroplet.

TABLE 5 Droplet Results for Four Non-Irradiated Petri Dishes. Water Dish1, Dish 2, Dish 3, Dish 4, Droplet Droplets Droplets Droplets DropletsSize 1, 2, 3 1, 2, 3 1, 2, 3 1, 2, 3 (microLiters): (Degrees) (Degrees)(Degrees) (Degrees) 25 34, 35, 37 31, 30, 29 33, 34, 34 34, 33, 34 5021, 22, 22 18, 19, 20 21, 20, 22 22, 23, 23 100  15, 16, 17.5 13.5, 14,14 14, 13, 12 14, 15, 14

TABLE 6 Droplet Results for Two 25 kGy Electron Beam Irradiated PetriDishes. Water Dish 1, Dish 2, Droplet Size Droplets 1, 2, 3 Droplets 1,2, 3 (microLiters): (Degrees) (Degrees) 25 44, 45, 44 44, 44, 45 50 25,26, 26 24 25, 26 100  17, 16, 16 17, 17, 16

TABLE 7 Droplet Results for Two 40 kGy Electron Beam Irradiated PetriDishes. Water Dish 1, Dish 2, Droplet Size Droplets 1, 2, 3 Droplets 1,2, 3 (microLiters): (Degrees) (Degrees) 25 44, 43, 45 38, 37, 37 50 28,29, 28 23, 24, 25 100  17, 18, 17 15, 14, 14

TABLE 8 Droplet Results for Three 55 kGy Electron Beam Irradiated PetriDishes. Water Dish 1, Dish 2, Dish 3, Droplet Size Droplets 1, 2, 3Droplets 1, 2, 3 Droplets 1, 2, 3 (microLiters): (Degrees) (Degrees)(Degrees) 25 39, 37, 38 40, 37, 38 37, 39, 40 50 25, 26, 26 26, 25, 2525, 24, 24 100  16, 15, 17 17, 16, 17 15, 14, 16

Only the non-irradiated Petri dishes showed droplet movement commencingwith a lesser/shallower elevation being sufficient for each size ofdroplet. This observation indicates greater hydrophobicity for thenon-irradiated Petri dishes. There was little incremental change withfurther irradiation beyond 25 kGy. With electron beam irradiation, themost decrease in Petri dish hydrophobicity (e.g., increased waterwettability and increased elevation angle to initiate water dropletmovement on the Petri dish surface) was obtained with an electron dosageof 25 kGy with very little additional change above that electron dosage.

Comparing elevation angle results for the various water droplet sizes onirradiated and non-irradiated dishes in Example 3 with thosecorresponding results in Example 2, it is apparent that the trending ofresults was similar in both Examples. However, the absolute elevationangles measured in Example 3 for the non-irradiated dishes wereapproximately twice as great as those in Example 2 (while the elevationangles for the irradiated dishes were approximately 50% greater). Theseresults may indicate that the polylactide surfaces of the dishes inExample 3 were somewhat more hydrophilic than those in Example 2 (moldedfrom different batches of resin).

Example 4: Cell Adhesion and Adherent Cell Culture

Experiments were conducted to investigate how mammalian cell growth on amixture of poly-L-lactide and poly-D-lactide is affected undercontrolled cell culturing conditions. Three well-established mammaliancell lines were employed in testing cell growth, substrate adhesion, andviability, including HEK293 suspension-adapted cells, HEK293 adherentcells, and COS adherent cells.

Cells were cultured in DMEM medium (Dulbecco's Modified Eagles Medium)supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and 100I.U./ml each of penicillin and streptomycin in a 37° C. incubator withan atmosphere of 5% CO₂ in air. As negative and positive culturing“controls”, the above three cell lines were cultured in either (i)sterile unmodified polystyrene Petri dishes suitable for bacteriologicaland yeast culture or (ii) sterile CelIBIND® surface-treated polystyrenePetri dishes from Corning, Inc. suitable for mammalian cell culture.Within 3-5 days incubation in the first set of dishes (i), no celladhesion was observed and microscopically all cells appeared dead. Inthe second set of dishes (ii) all cells appeared viable and the HEK293and COS adherent cells were attached and growing in monolayers. Theseresults confirm that modification of conventional thermoplastic surfacessuch as polystyrene is needed for anchorage and growth of multiplevarieties of mammalian cells

Next, 90 mm diameter Petri dishes that were injection-molded by EcNowTech, Inc. (Albany, Oreg.) using melt blends that combined approximately1% poly-D-lactide and 99% poly-L-lactide were tested. These enantiomerpolymer-blend Petri dishes (abbreviated “EP blend” dishes) werethermally annealed to promote the crystallization of the mixture ofPLA/PDA and to provide thermal stability. Depending upon the annealingconditions of the PLA/PDA mixture, including but not limited totemperature and dwell time, thermal stabilities exceeding at least 60°C., or at least 65° C., or at least 70° C., or at least 75° C., or atleast 80° C., or at least 85° C., or at least 90° C. were achieved.Thermal stabilities were evaluated by incubating the EP blend dishes inair for 30 minutes at the testing temperature and observing whether anyvisible warping of the flat bottom of the dishes had occurred. Followingmolding, the EP blend dishes were packaged in plastic film sleeves andsent out for sterilization using two different methods: One group wasirradiated using cobalt 60 gamma irradiation (@25 kGy dosage) and twogroups were irradiated using electron beam irradiation (@25 kGy dosageand @55 kGy dosage at 1 MeV electron energy).

The above 3 groups of sterilized EP blend dishes were biologicallytested using the same three cell lines described above cultured in thesame DMEM medium (see above). Remarkably, in the absence of any addedsurface treatment or modification of the EP blend PLA/PDA surfaces, whenthe above HEK293 adherent cells were seeded on the bottom interiorsurfaces of all three sterilized groups of the EP blend PLA/PDA dishes,the cells settled to approximately 25% confluence and grew steadily asmonolayers to approximately 80% confluence within 72 hours. Nodifferences in growth rates, cellular adhesion or cellular morphologiescould be discerned among the dishes that had been sterilized using thethree different protocols. Incubation with trypsin allowed easydetachment of the HEK293 adherent cells in all PLA/PDA dishes forsubsequent passage. By comparison, the COS adherent cells that had beenseeded in somewhat larger cellular patches, reached over 100% confluencein less than 72 hours. These somewhat overgrown cells were more tightlyadhered to the substrate surface than the HEK293 adherent cells and weremore difficult to detach even with trypsin incubation and vigorousagitation. Beneficially, however, with a lower seeding density thesesame cells grown for 72 hours without overgrowth were easily detachedwith trypsin incubation and successfully passaged. Finally, thesuspension-adapted HEK293 cells grew at approximately the same rate over72 hours as the HEK293 adherent cells, reaching approximately 60%confluence with more loosely adhered cells that were efficientlydetached with trypsin, and with the balance of viable cells in suspendedpatches with no apparent loss in cellular viability.

For comparison purposes, the same three cell lines were simultaneouslyand successfully cultured on CeIIBIND® surface-treated polystyrene Petridishes (Corning, Inc.) and grew with cellular morphologies and growthrates that were indistinguishable from the growth patterns on the EPblend PLA/PDA dishes. By contrast, failure of these same three celltypes to adhere and/or grow on plain (unmodified/uncoated) polystyrenePetri dish surfaces that are suitable for bacteriological and yeastculture was previously described above.

Example 5: Bacterial and Yeast Cell Culture

Regarding the ability to carry out normal diagnostic bacterial cellculturing on gelled nutrient agar surfaces in the same EP blend PLA/PDAdishes as described in Example 1, the growth of E. coli cells wasmonitored in PLA/PDA dishes that had been cobalt 60-irradiated andsterilized (25 kGy dosage). Approximately 20 ml of Luria Broth (LB)bacterial growth medium (1% tryptone, 1% NaCl, and 0.5% yeast extract inwater) with or without ampicillin (amp) antibiotic (100 micrograms perml) and further containing 2% dissolved agar was poured into each dishat a temperature of 55° C. After agar solidification, both amp-sensitiveand amp-resistant strains of E. coli were streaked out and incubated at37° C. As a “positive control” a dish containing LB agar lacking anyantibiotic allowed both amp-sensitive and amp-resistant cells to grownormally overnight. In dishes containing LB agar+antibiotic, theamp-resistant cells grew but not the amp-sensitive cells as anticipated.

Regarding the ability to carry out normal diagnostic yeast cellculturing on gelled nutrient agar surfaces in the same EP blend PLA/PDAdishes, the growth of S. cerevisiae yeast cells was monitored in thesePLA/PDA dishes that had been cobalt 60 gamma-irradiated and sterilized(25 kGy dosage). Approximately 20 ml of YEPD medium (2% Bacto peptone,1% yeast extract and 2% dextrose in water) and further containing 2%dissolved agar was poured into each PLA/PDA dish at a temperature of 55°C. After agar solidification, S. cerevisiae yeast cells were streakedout on the medium and incubated overnight at 30° C. The yeast cells grewnormally and rapidly on the agar-gelled YEPD surface. Similarly, normalgrowth rates were observed for S. cerevisiae cells streaked out onminimal defined media contained in the same EP blend PLA/PDA dishes.Finally, microscopic examination of S. cerevisiae cells that had beenplated out and incubated at 30° C. on a yeast sporulation mediumsupplemented with 1% potassium acetate and formulated to inducesporulation (also contained in identical EP blend PLA/PDA dishes) showedthat the yeast cells experienced normal rates of sporulation withformation of normal four spore tetrads.

As used herein, “consisting essentially of” allows the inclusion ofmaterials or steps that do not materially affect the basic and novelcharacteristics of the claim. Any recitation herein of the term“comprising”, particularly in a description of components of acomposition or in a description of elements of a device, can beexchanged with “consisting essentially of” or “consisting of”.

What is claimed is:
 1. A cell culture container consisting essentiallyof a polylactide polymer material, the material consisting of (100−X) wt% of poly-L-lactide and X wt % of poly-D-lactide, wherein X≤10, wherein100 wt % represents the total weight of polylactide polymers in thematerial, and wherein the cell culture container is formed from thepolymer material by a forced flow molding method and has been sterilizedby gamma irradiation or electron beam irradiation, thereby increasingthe hydrophilicity of a cell growth surface of the container compared tothe cell growth surface without said irradiation.
 2. The cell culturecontainer of claim 1, wherein the forced flow molding method is selectedfrom the group consisting of blow molding, rotational blow molding,injection blow molding, injection stretch blow molding, extrusionmolding, extrusion molding followed by thermoforming, thin-wallinjection molding, micro injection molding, and gas-assisted injectionmolding.
 3. The cell culture container of claim 1, wherein the containeris biodegradable.
 4. The cell culture container of claim 1, wherein thecontainer is compostable.
 5. The cell culture container of claim 1,wherein the material is transparent.
 6. The cell culture container ofclaim 1 that is viability-sustaining for a live culture ofanchorage-dependent cells.
 7. The cell culture container of claim 1 thatis viability-sustaining for a live culture of suspension-growing cells,suspension-adapted cells, anchorage-dependent cells, and cells growingon a gelled culture medium.
 8. The cell culture container of claim 1,wherein the container is configured as a container selected from thegroup consisting of a Petri dish, a cell culture flask, a multi-wellplate, and a roller bottle.
 9. The cell culture container of claim 1,including one or more cell growth structures selected from the groupconsisting of channels, lattices, matrices, webs, sponges, fibers,scaffolds, and beads; wherein said one or more cell growth structuresconsist essentially of a polylactide polymer material, the materialconsisting essentially of (100−X) wt % of poly-L-lactide and up to X wt% of poly-D-lactide, wherein X≤10, and wherein 100 wt % represents thetotal weight of polylactide polymers in the mixture.
 10. The cellculture container of claim 1, wherein said cell growth surface of thecontainer is devoid of any surface coating or chemical or physicalsurface modification other than by gamma irradiation or electron beamirradiation.
 11. The cell culture container of claim 1, wherein X=0. 12.The cell culture container of claim 1, wherein said irradiation is at adose in the range from about 25 kGy to about 55 kGy.
 13. The cellculture container of claim 1, wherein the increase in hydrophilicity isassociated with an increase in tilt angle of a water droplet on saidcell growth surface after said gamma irradiation or electron beamirradiation.
 14. A cell growth structure consisting essentially of apolylactide polymer material consisting of (100−X) wt % ofpoly-L-lactide and X wt % of poly-D-lactide, wherein X≤10, and whereinthe cell growth structure is formed by a forced flow molding method andhas been sterilized by gamma irradiation or electron beam irradiation,thereby increasing the hydrophilicity of a cell growth surface of thecell growth structure compared to the cell growth surface without saidirradiation.
 15. The cell growth structure of claim 14, wherein theforced flow molding method is selected from the group consisting of blowmolding, rotational blow molding, injection blow molding, injectionstretch blow molding, extrusion molding, extrusion followed bythermoforming, thin-wall injection molding, micro injection molding, andgas-assisted injection molding.
 16. A method of making a cell culturecontainer, the method comprising: (a) heating a polylactide polymermaterial consisting of (100−X) wt % of poly-L-lactide and X wt % ofpoly-D-lactide, wherein X≤10 to a molding temperature, and wherein 100wt % represents the total weight of polylactide polymers in the mixture;(b) subjecting the material to a molding method comprising forced flowmolding to form a structure having a shape of said cell culturecontainer; (c) cooling the structure; (d) optionally annealing thestructure for an annealing time at an annealing temperature; and (e)sterilizing the container obtained from step (c) or step (d) with gammairradiation or electron beam irradiation; whereby said cell culturecontainer is obtained.
 17. The method of claim 16, wherein the forcedflow molding method is selected from the group consisting of blowmolding, rotational blow molding, injection blow molding, injectionstretch blow molding, extrusion molding, extrusion followed bythermoforming, thin-wall injection molding, micro injection molding, andgas-assisted injection molding.
 18. A method of culturing cells, themethod comprising: (a) providing the cell culture container of claim 1;(b) adding cells and a culture medium to the container; and (c)incubating the container, cells, and culture medium for an incubationtime and at an incubation temperature such that the cells are viablysustained and optionally reproduce.
 19. The method of claim 18, furthercomprising after step (c): (d) allowing the container to be biodegradedand/or composted.
 20. The method of claim 19, wherein the cells comprisean anchorage-dependent cell type, and wherein the anchorage-dependentcell type adheres to said growth surface of the cell culture container,wherein said growth surface is devoid of any surface coating or chemicalor physical surface modification other than by gamma irradiation orelectron beam irradiation.